Specific Aim 1: Discovery of A New Pathway for the Activity-Dependent Plasticity through Axonal Transport Neuronal transport includes the intracellular trafficking route for membranous protein carriers from the soma to nerve terminals where they deliver cargos for synapse formation. The contents of these transport packets include protein components of SV and AZs, exocytotic machinery, channels, and adhesion molecules. Cargos must be attached to their transport motors with a high degree of specificity to preserve cargo identity and targeted trafficking. However, the mechanism underlying motor-cargo interactions remains unresolved. The SNARE protein syntaxin-1, a key component of SV fusion machinery, is transported to the plasma membrane in cargos. We identified a novel syntaxin-binding and kinesin-1 motor (KIF5)-associated protein named syntabulin (1, 2). Our studies with loss-of-function analysis established that syntabulin is an adaptor capable of conjoining syntaxin-1 and KIF5 motors, thereby mediating transport of syntaxin-1 to neuronal processes. Remodeling of pre-existing synapses and the formation of new synapses play an important role in the various forms of synaptic plasticity of complex neuronal networks. Previously identified mechanisms underlying activity-dependent synaptic plasticity include activation of transcriptional factors, new protein synthesis, and reorganization of the actin filaments at synapses. Thus, efficient and targeted axonal transport of newly synthesized synaptic components to presynaptic boutons would be critical in response to neuronal activity. However, the contribution of the microtubule-based axonal transport to the activity-induced formation of new synapses are unknown. Since syntaxin-1 is a component of the AZ precursor cargos, further characterization of syntabulins role in neuronal trafficking will contribute to understanding the molecular mechanisms of the axonal delivery of presynaptic components. Our ongoing research reveals that syntaxin-1, syntabulin, and KIF5 comprise the transport machinery critical for anterograde axonal transport of the AZ precursors and contributes to presynaptic assembly (3). Knockdown of syntabulin or disruption of the syntaxin-1-syntabulin-KIF5B complex impairs the anterograde transport of AZ components out of the soma and reduces the axonal densities of SV clusters and FM4-64 loading. Furthermore, syntabulin loss-of-function results in a reduction in both the amplitude of postsynaptic currents and the frequency of asynchronous quantal events, and abolishes the activity-induced recruitment of new AZ components into the axons and subsequent co-clustering with SVs. Consequently, syntabulin loss-of-function blocks the formation of new presynaptic boutons during activity-dependent synaptic plasticity. These studies establish for the first time that a kinesin motor-adaptor complex is critical for the anterograde axonal transport of AZ components, thus contributing to activity-dependent presynaptic assembly during neuronal development. To explore the function of syntabulin in vivo, we will generate the conditional KO mice with functional disruption of the syntabulin gene. Phenotype analysis of the KO mice and the mutant neurons will clarify the molecular details of how this protein mediates the trafficking of the AZ precursor vesicles essential for presynapse assembly and synaptic plasticity. Specific Aim 2: Identification of An Essential Role of Snapin in Synchronizing Fast Neurotransmitter Release Information coding in the brain depends on the timing of action potentials, which is influenced by integration of unitary excitatory inputs. The size and shape of excitatory postsynaptic currents (EPSCs) are two decisive factors in tuning the temporal and spatial precision of spiking and can be modulated by synaptic vesicle (SV) fusion process. Ca2+-triggered neurotransmitter release depends on the presence of a pool of primed release-ready SVs, which determines the release probability of a synapse. The priming step corresponds to assembly of the SNARE complex in which synaptobrevin interacts with SNAP-25 and syntaxin-1 to form a metastable structure before fusion. Maturation of SVs into a release-ready state requires synaptotagmin I (Syt I), a Ca2+ sensor of fast neurotransmission. Accurate assembly of Syt I-SNARE fusion machineries is critical for the precise timing of fast release. Ca2+-dependent and independent interactions between Syt I and SNAREs suggest that before the Ca2+ trigger, a loose pre-fusion Syt I-SNARE complex is assembled during the priming process. Ca2+ influx sensitizes the Ca2+ sensor Syt I and induces its subsequent tight coupling to the SNARE complex. While much attention in the past decade has been given to the SNARE-regulatory proteins in studying SV release probability and short-term plasticity, our understanding of the molecular mechanisms that govern the tuning of EPSC shape is largely lacking. We initially identified Snapin as a SNAP-25-binding protein that enhances the association of Syt I with the SNAREs (4-6). Using snapin knockout mice, we demonstrated that Snapin modulates fast exocytosis of large dense-core vesicles in chromaffin cells (7). Deletion of snapin leads to a reduced amount of Syt I-SNARE complex in mouse brain. We recently characterized the function of Snapin in synchronizing SV fusion at central synapses (8). By recording synaptic transmission between cultured cortical neurons from snapin-deficient mice, we found that snapin mutant neurons exhibit EPSCs with multiple peaks and fail to follow sustained firing under high-frequency stimulations. Re-introducing snapin into the mutant presynaptic neurons effectively accelerates EPSC kinetics to the greater extent found in (+/+) neurons by boosting the synchronicity of SV fusion. The marked increase in rise/decay time and synaptic delay time observed in snapin-deficient neurons changes the shape of the EPSC and impairs both synaptic efficacy and precision. At snapin-deficient nerve terminals, SVs are likely heterogeneously primed due to the unfavorable or unstable association of Syt I with the metastable SNARE complex before the Ca2+ sensing. It leads to two defects: (1) fewer fusion competent vesicles, and hence decreased size of EPSCs;and (2) fewer vesicles undergoing synchronized fusion within a narrow time window during excitation-secretion coupling. Thus, our studies reveal the role of Snapin as a unique synchronizer of calcium-triggered SV fusion at central synapses. Papers published from the lab related to the projects: 1. Qingning Su*, Qian Cai*, Claudia Gerwin, Carolyn L. Smith, Zu-Hang Sheng (2004). Nature Cell Biology 6, 941-953. 2. Qian Cai, Claudia Gerwin, and Zu-Hang Sheng. (2005). Journal of Cell Biology 170, 959-969. 3. Qian Cai, Pingyue Pan, and Zu-Hang Sheng. (2007). Journal of Neuroscience 27, 7284-7296. 4. Jeffrey M. Ilardi, Sumiko Mochida, and Zu-Hang Sheng (1999). Nature Neuroscience 2, 119-124. 5. Milan G. Chheda, Uri Ashery, Pratima Thakur, Jens Rettig, and Zu-Hang Sheng (2001). Nature Cell Biology 3, 331-338. 6. Pratima Thakur, David R. Stevens, Zu-Hang Sheng and Jens Rettig (2004), Journal of Neuroscience 24, 6476-6481. 7. Jin-Hua Tian, et al (2005). Journal of Neuroscience 25, 10546-10555. 8. Ping-Yue Pan, Jin-Hua Tian and Zu-Hang Sheng (2009). Neuron 61, 412-424.